The goal of this project is to investigate the role of L-arginine as substrate for nitric oxide synthesis in the pathophysiology of severe falciparum malaria. Globally, malaria causes more morbidity and mortality than any other parasitic disease with most deaths occurring in African children. Nitric oxide (NO) metabolites have been shown to be low in severe malaria. Studies performed by our group have shown that plasma arginine concentrations are markedly depressed in Tanzanian children with cerebral malaria when compared to healthy control children and children with uncomplicated malaria. Furthermore, arginine levels less than 45 mu-M in cerebral malaria were predictive of mortality. We hypothesize that low arginine in severe malaria may be due to one or a combination of the following: 1. reduced intestinal uptake, 2. reduced endogenous renal synthesis, 3. increased consumption of arginine in the urea cycle, or 4. increased catabolism of arginine by extrahepatic arginase induced in mononuclear cells and other cell types. These hypotheses will be investigated by three different approaches. The first approach will be to determine plasma levels of amino acids associated with the uptake, biosynthesis and catabolism of arginine, and levels of other essential and non-essential amino acids. Plasma samples will be analyzed using HPLC with pre-column derivatization and fluorescence detection. The second approach will be to determine the flux and metabolic fate of arginine in patients with severe malaria and to define how they differ from healthy controls. This will be accomplished by infusing non-radioactive tracers of arginine, citrulline, ornithine and leucine and measuring the metabolic byproducts of these tracers. HPLC separation coupled to mass spectrometric analysis of heavy isotope concentration in collected samples will be used. The third approach will be to directly determine the transcription of enzymes important for the uptake, biosynthesis, and catabolism of arginine. Collected peripheral blood mononuclear cell extracts will be analyzed using real time PCR. An enzymatic assay to measure arginase activity in mononuclear cell extracts will also be used. This project is essential for the overall goal of further defining the pathophysiology of severe malaria. The knowledge gained will facilitate the development of disease modifying strategies (e.g. arginine or other amino acid infusion) to reduce the morbidity and mortality of falciparum malaria.